The Wnt/β-catenin cell signaling pathway is implicated in a variety of developmental processes including stem cell maintenance and growth, cellular differentiation, cell growth, oncogenesis and disease pathogenesis (Kirikoshi et al, Int. J. Oncol. 19:767-771 (2001); Munroe et al, Proc. Natl. Acad. Sci. USA 96:1569-1573 (1999); Reya and Clevers, Nature 434:843-850 (2005); Sher et al, FEBS Lett. 522:150-154 (2003)). The activation and regulation of the Wnt/β-catenin pathway therefore appears to be critical for tissue homeostasis, regeneration and repair. The “canonical” Wnt cell signaling pathway has as its central player, the cytosolic protein β-catenin (FIG. 1). When Wnt receptors are not engaged, the level of cytosolic β-catenin is kept low through the action of an intracellular complex, known as the “destruction complex,” composed of the tumor suppressor proteins axin and adenomatous polyposis coli (APC) and the serine kinase protein glycogen synthase 3β (GSK3β). The constitutive kinase activity of the destruction complex on β-catenin results in the targeted proteosomal degradation of phosphorylated β-catenin. Binding of Wnt to the receptor proteins LRP5 and/or LRP6, members of the LDL receptor family, and Frizzled (FZD), a serpentine receptor, induces phorphorylation-dependent binding of Axin to the LRP6 cytoplasmic tail and recruitment of the cytoplasmic protein Dishevelled (Dvl) to the cytoplasmic tail of FZD, which together lead to the inactivation of the β-catenin destruction complex. As a consequence, β-catenin accumulates in the cytoplasm and translocates to the nucleus where it is thought to interact with members of the lymphoid enhancer factor (LEF)/T-cell factor (TCF) family of transcription factors and activate target gene expression.
The Wnt coreceptors LRP5/6 are modulated by the secreted ligands Dkk1, Dkk2 and SOST/Sclerostin, a ligand for LRP5/6 and a Wnt signaling inhibitor. Interaction of SOST or Dkk1/2 wth LRP5/6 antagonizes Wnt/β-catenin signaling. Dkk1 is a high affinity ligand for LRP5/6 and disrupts the formation of the FZD-LRP complex. Dkk1 also binds Kremen-1 and -2 which are single-pass transmembrane proteins that cooperate with Dkk1 to inhibit Wnt-FZD-LRP6 function. Upon binding of Dkk1 to LRP6 and Kremen-1, receptor complex internalization occurs thereby dampening the Wnt signal due to a decrease of the Wnt coreceptors available for signaling indicating that the cell surface levels of LRP5/6 may limit cellular responses to Wnt ligands (reviewed in He et al, Development 131:1663-1677 (2004) and Semënov et al, J. Biol. Chem. 283:21427-21432 (2008)).
An area in which Wnt signaling has been implicated is the regulation of bone mass in homeostasis and bone disease. Bone mass appears to be influenced by the balance achieved between bone forming cells (osteoblasts) and bone resorbing cells (osteoclasts). Mutations in LRP5 and LRP6 receptors have been reported that either decrease or increase bone density, indicating that the level of Wnt and/or LRP5/6 signaling is critical for maintaining normal bone homeostasis. Consistant with these findings, it has been reported that elevated levels of the LRP5/6 inhibitor Dkk1 in diseases such as rheumatoid arthritis and multiple myeloma, result in osteolytic bone lesions, which can be reversed by Dkk1 antagonists, indicating that Dkk1 may be a regulator of bone density (reviewed in Krishnan et al, J Clin Invest 116:1202-1209 (2006)).
Several studies have also implicated the Wnt signaling pathway in tissue homeostasis and repair in a variety of systems including intestinal, epidermal, and hematopoietic systems. In the intestine, continuous renewal of absorptive epithelium is driven by proliferation of stem cells residing in the intestinal crypts. Current evidence suggests that the Wnt signaling cascade is important in controlling stem cell function in the intestinal crypt since deletion of the β-catenin-dependent transcription factor TCF4 in mice results in depletion of intestinal crypts and loss of intestinal function (Korinek et al., Nat Genet. 19:379-83 (1998); Barker and Clevers, Nature Rev. 5:997 (2006)). Similarly, overexpression of Dkk1 in the intestine in transgenic mice or in mice injected with adenovirus expressing Dkk1, resulted in a complete loss of crypts in adult mice (Kormek et al, Nature Gen. 19:1-5 (1998); Kuhnert et al., Proc Natl Acad Sci USA. 101:266-71 (2004)). Intestinal diseases, such as inflammatory bowel disease, ulcerative colitis and radiation- or chemotherapy-induced mucositis, are associated with intestinal lesions and loss of intestinal absorptive epithelium, suggesting that modulation of Wnt signaling in intestinal crypts could have therapeutic benefit in treating such diseases.
A similar mechanism of Wnt signaling regulating stem cell function, tissue homeostasis and repair is found in the skin. Hair follicle density and the hair cycle are regulated by Wnt-dependent hair follicle epithelial stem cells (van Genderen et al, Genes Dev. 8:2691-2703 (1994); Lo Celso et al, Development 131:1787-1799 (2004)). Interestingly, the LRP5/6 inhibitor Dkk1 is expressed adjacent to hair follicle buds and over-expression of Dkk1 reduces hair follicle density, indicating that the level of LRP/Wnt signaling is important for regulation of hair follicle density and that Dkk1 may be a regulator of this process (Sick et al., Science. 314:1447-50 (2006)). Recently it was shown that hair follicle stem cells contribute to re-epithelialization during wound healing (Ito et al, Nat. Med. 11:1351-4 (2005)), indicating that modulation of Wnt signaling in hair follicle stem cells could be beneficial for wound repair.
In addition to the examples described above, Wnt signaling has also been shown to be important for regulation of stem cells in other tissues and organs, including hematopoietic stem cells (Reya et al, Nature 423:409-414 (2003); Xu et al., Nature Immunol. 4:1177-1182 (2003)), neuronal progenitor stem cells (Zecher et al, Dev. Biol. 258:406-418 (2003)), and even embryonic stem cells (Sato et al, Nature Med. 10:55-63 (2004)) suggesting that modulation of Wnt signaling could also have therapeutic benefits in these systems.
Thus, molecules that modulate Wnt signaling can be useful targets for a broad range of conditions where proliferation, differentiation, tissue regeneration and repair are important to disease processes. The present invention provides anti-LRP6 antibodies that enhance LRP6 activity and antagonize Dkk1 activity for treatment of diseases such as, but not limited to bone disorders such as osteoporosis and osteolytic lesions caused by osteoarthritis and multiple myeloma as well as gastrointestinal disease and wound healing.